Nanoparticles in Measurement Science - Analytical Chemistry (ACS

Review pubs.acs.org/ac

Nanoparticles in Measurement Science Francis P. Zamborini,*,† Lanlan Bao,† and Radhika Dasari‡ †

Department of Chemistry, University of Louisville, Louisville, Kentucky 40292, United States Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, United States

‡

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CONTENTS

Spectroscopic Analysis Surface Plasmon Resonance (SPR) Spectroscopy Localized Surface Plasmon Resonance (LSPR) Spectroscopy Fluorescence Spectroscopy Surface-Enhanced Raman Spectroscopy (SERS) Mass Spectrometry Electronic Detection Introduction Applications Separations Introduction Applications Summary and Future Outlook Author Information Corresponding Author Biographies References

spectroscopy, although they have been used in these techniques. Electronic based detection methods include electrochemical detection and chemiresistive sensors. Because the literature on electrochemical detection methods using NPs is vast and too large to cover in this review, we only discuss it briefly and focus more on chemiresistive sensors. In the section on separations, we review various chromatographic and electrophoretic separations involving NPs. Several different sensor technologies exist that utilize the benefits of NPs, which includes piezoelectric sensors, such as cantilever sensors, the quartz crystal microbalance (QCM), and the surface acoustic wave (SAW) device. While interesting and important, we did not include these examples in this review. This review focuses on the use of NPs to improve the detection of chemical and biochemical analytes by the various analytical techniques and sensor strategies rather than analysis of the NPs themselves. This review was limited to 250 references, making it impossible to cover all of the literature in this area over the past 2 years. We chose examples that cover a wide range of methods and those that represent new approaches, as opposed to those that used a similar approach reported several years back but with a new type of analyte. We apologize to the authors of important publications that we may have excluded due to our limitations.

541 541 543 552 561 566 567 567 567 570 570 570 571 572 572 572 572

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his review describes recent advances in the use of nanoparticles (NPs) for analytical measurements. Over the past decade, the field of nanotechnology has undergone tremendous developments, resulting in new procedures for the controlled synthesis of a wide variety of nanoscale materials and measurement of their unique properties. These properties, including optical, electronic, magnetic, chemical, mechanical, and catalytic, can often be tuned by the size, shape, and composition of the nanostructure. These unique properties and their generally high surface-to-volume ratio has led to their use in several analytical applications. With the widespread use of nanostructures for analytical applications, it is important to review some of the most recent and exciting advances. This review covers the time period of January 2010 to November 2011. For this review, we define NPs as any material with at least one dimension in the 1−100 nm range. The shape may vary (it does not have to be spherical) and the materials include metals, semiconductors, polymers, and carbon based materials. NPs have been used for various applications in the traditional disciplines of analytical chemistry, including spectroscopy, electronic detection, and separations, as well as in various sensor technologies. In the spectroscopy area, we review the use of NPs in surface plasmon resonance (SPR) spectroscopy, localized surface plasmon resonance (LSPR) spectroscopy, fluorescence spectroscopy, surface-enhanced Raman spectroscopy (SERS), and mass spectrometry. We did not include the use of NPs in nuclear magnetic resonance (NMR) spectroscopy, magnetic resonance imaging (MRI), or infrared (IR) © 2011 American Chemical Society

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SPECTROSCOPIC ANALYSIS Surface Plasmon Resonance (SPR) Spectroscopy. Introduction. Surface plasmon resonance (SPR) is a versatile method for detecting changes in the refractive index occurring on thin metal films, usually Au, as a result of recognition events or chemical reactions.1 By detecting a small refractive index change at the metal−analyte interface, the information of the molecular interactions can be obtained by measuring the optical characteristics (intensity, phase, and polarization) of light reflected from the optical setup, usually using a Kretschmann configuration.2 SPR was first proposed for the direct monitoring of specific antibody−antigen interactions in the 1980s.3 Since then, the technology has grown rapidly and resulted in a vast array of platforms available commercially or in researchlaboratory settings.4 SPR sensing is a label-free and sensitive technique for the detection of molecular interactions. The advantages of this technique are well stated in a recent paper published by Gao et al.5 In SPR sensors, changes in the plasmonic resonance signals at a thin metal film are strongly dependent on the refractive index of the medium.5 The sensitivity of the method is limited Special Issue: Fundamental and Applied Reviews in Analytical Chemistry Published: December 7, 2011 541

dx.doi.org/10.1021/ac203233q | Anal. Chem. 2012, 84, 541−576

Analytical Chemistry

Review

Figure 1. (A) The SPR based detection system. (B) The SPR response curve before (black) and after (green) the analyte binding. (C) SPR sensorgram.

small molecules or ions,1 such as Hg2+.6 Third, we review Au NP enhanced SPR imaging (SPRI) for biosensing applications.10,14 Finally, we describe magnetic NPs as signalenhancement labels for SPR sensing.8,15,16 Gold Nanoparticle Enhanced SPR Biosensing. Au NPs have been conjugated to a secondary biomolecular probe in a sandwich assay with the metal surface to enhance the sensitivity and detection limit of SPR sensors.11−13 Kim et al.11 demonstrated ultrasensitive detection of a protein biomarker, immunoglobulin E (IgE), using biofunctionalized Au NPs to form the sandwich complexes with an Au surface as the SPR sensor. Two sandwich assay complexes were created via the adsorption of IgE onto (i) surface bound anti-IgE followed by the adsorption of IgE-aptamer coated Au NPs and (ii) an IgEaptamer surface before the adsorption of anti-IgE coated Au NPs. The shift of the resonance angle was recorded to monitor the interactions of the analyte with the surface. Both methods are capable of IgE detection down to 1 fM, which is a 106 improvement over conventional SPR measurements. The same research group demonstrated a sandwich assay for the detection of IgE using anti-IgE labeled Au nanorods.12 Au nanorods further increased the detection limit to attomolar concentration, which is 103 times more sensitive compared to the Au NP assay. Although Au NP labeled SPR sandwich assays show high sensitivity for biosensing applications, the sensitivity of the system decreases when the probe size is smaller than the target molecule.5 The attachment of the big target molecule on the probe blocks the neighboring probes from interaction with the target. A dendrimer-encapsulated Au NP was used instead of a bare Au NP to address the problem of steric hindrance as described by Frasconi et al.17 This makes the probe big and gives the probe enough space to interact with the target. This assay detected insulin at a limit of 0.5 pM. Instead of using a sandwich assay for NP-enhanced SPR sensing, modification of the metal film with metallic NPs can also lead to an amplified SPR signal. Au NPs can be deposited directly on the surface to form a nanocomposite film,5 or one can chemically cross-link the Au NPs on the Au electrode to prepare the modified SPR surface.1 Gao et al.5 produced ultrathin Au/Al2O3 nanocomposite films via a radio frequency

by the magnitude of the refractive index change at the metal surface and the minimum SPR shift that is detectable by the instrument as a result of recognition events occurring between a surface-bound receptor and analyte of interest (see Figure 1).6 The surface modification leading to the immobilization of molecular recognition elements specific to a target analyte is therefore of great importance in SPR detection.7 The reflected light is measured from the metal−solution interface as shown in Figure 1A. The surface plasmon of the metal film that is excited by the p-polarized light causes a strong decrease in reflectivity at a specific angle of the incident light, which depends on the refractive index of the medium. A solution containing analyte flows over the surface, and upon recognition and binding of analyte to the Au surface, the refractive index changes. As a result, the angle of minimum reflection shifts as shown in Figure 1B. In practice, the angle of minimum reflectance or the reflected light intensity may be plotted as a function of time when detecting analyte. If the angle of minimum reflectance is chosen and the analyte binds, the change in refractive index will lead to an increase in reflected light intensity, since the incidence angle will no longer be at the angle of minimum reflectance. This leads to a sensorgram similar to that shown in Figure 1C. The sensorgram shows the dynamics of the binding and desorption of analyte on the Au surface. A bigger difference in refractive index change upon analyte binding leads to bigger angle shifts and larger intensity changes. The signal decreases back to the baseline upon dissociation of the analyte from the receptor. SPR technology has some sensitivity limitations for the detection of target with very low concentration in real samples or for small molecule-detection.7,8 There are various strategies, including metal nanoparticle (NP) labels, which offer excellent signal amplification.9 Coupling between the localized surface plasmon of metal NPs and the surface plasmon wave of the metal film was found to increase the shift in the SPR spectrum.6 In this section, we review recent literature on the use of NPs for signal amplification in SPR sensing. We review biosensing applications first, since this is the most common application for SPR. Au NPs, including spherical and rod shaped NPs are the most common NPs used to enhance SPR signals.10−14 The second part of this section is NP enhanced SPR sensing of 542



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MNP-labeled secondary antibody to the thrombin bound to antithrombin on the surface. The sensor exhibited a detection limit of 0.017 nM and high specificity. The same group demonstrated the detection of adenosine using Fe3O4 MNPantiadenosine aptamer conjugates for signal amplification.15 Using a similar idea, Pollet et al.16 demonstrated the detection of peanut allergen with a magnetic nanobead enhanced optical fiber SPR for the first time. The detection limit was 9 μg/mL (2 orders of magnitude better than SPR fiber optic sensors without the NP label) with a linear range between 0.1 and 2 μg/mL. Future Directions. NP-based SPR sensors have shown excellent specificity and sensitivity toward biomolecules, small molecules, and ions at low concentration. The sensitivity can be further improved by using NPs with different sizes, shapes, and composition. Future experiments will likely explore these different variables to further maximize signal amplification and optimize these sensors. Localized Surface Plasmon Resonance (LSPR) Spectroscopy. Introduction. Localized surface plasmon resonance (LSPR) refers to the collective oscillation of the conducting electrons of metal NPs when their frequency matches that of the incident electromagnetic radiation. It is one of the characteristic optical properties of metal NPs, such as Au and Ag,21 and serves as the basis for SPR (although not localized) and surface-enhanced Raman spectroscopy (SERS). A strong absorption band(s) or increased scattering intensity of the radiation occurs at certain wavelengths for the metal NPs as a result of this phenomenon.22 Acording to Mie theory, LSPR of the NP is mainly related to the NP size, shape, composition, interparticle distance, and dielectric constant (refractive index) of the surrounding medium.21,23−27 The latter property is the basis of biological and chemical LSPR sensors. There are five main different types of LSPR-based sensing strategies using metal NPs, including (1) detection of their LSPR absorbance/scattering or color using them as labels, (2) bulk refractive index sensing of the medium by direct detection of the LSPR wavelength shift, (3) detection of local analyte binding by direct detection of the LSPR wavelength shift, (4) detection of a LSPR wavelength shift by analyte-induced metal NP aggregation, and (5) detection of analyte induced distance changes in linked metal NPs (also known as plasmonic rulers). Figure 2 shows a schematic representation of the different sensing strategies. In this section of the review, we summarize the recent literature on NP-based LSPR sensing. First, we describe metal NPs as labels, as this is the simplest form of sensing. Second, we describe bulk refractive index sensing, including a recent protocol to improve the refractive index sensitivity and applications in gas and vapor sensing. Third, we describe sensing based on the refractive index change upon local binding of analyte on the nanostructure, which offers label-free sensing. Fourth, we summarize recent sensing studies based on metal NP aggregation, including metal ion sensing and biosensing. Fifth, we discuss the use of plasmon rulers to detect analyte or molecular conformational changes. Finally, we discuss LSPR imaging and other applications that do not fit into the main strategies. There are a few excellent reviews in 2010 and 2011 that summarize some of the nanostructure-based LSPR sensors. For example, Mayer et al.28 extensively described the analytical theory of LSPR, basics of LSPR sensing, details of the factors that affect the sensitivity, and literature on biological and biomedical LSPR assays. Sau et al.29 published a review about

magnetron cosputtering method. This film was deposited onto the Au surface for SPR sensing. The deposited film showed high stability in different solutions (ethanol, water, biotin thiol solution, and pH 7 buffer solution) and the interparticle distance could be tuned by changing the surface coverage. These features are extremely important when the probe size is smaller than the target molecule, since the Au NPs are well distributed from each other. Gold Nanoparticle Enhanced SPR Small Molecule Sensing. It is difficult for conventional SPR to detect small molecules and ions since they usually have smaller refractive index changes. Using NPs as a signal enhancing method makes SPR possible for sensing small molecules and ions. For example, Wang et al.18 detected Hg2+ in an aqueous solution using Au NPs in an SPR sandwich assay. They immobilized a mercury-specific oligonucleotide probe on the Au film, which specifically interacted with Hg2+ from the analyte solution. Then, Au NPs coated with partially cDNA strands were used to form the sandwich assay and amplify the signal. This method showed excellent specificity in the presence of other metal ions. The 5 nM detection limit is 2 orders of magnitude better than the sensor without NP amplification. Willner and co-workers described the electropolymerization of a bis-aniline-cross-linked Au NP composite film on an Au electrode for ultrasensitive SPR detection of trinitrotoluene (TNT).19 Recently, they extended the application of the system to the detection of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) with a detection limit of 12 fM.1 Gold Nanoparticle Enhanced SPR Imaging for Biosensing. In SPR imaging (SPRI), the reflectivity change is determined by measuring the SPR signal at a fixed angle of incidence before and after selective molecular adsorption across a fixed surface. SPRI has been increasingly used for label-free detection of DNA, in which the cDNA sequence is covalently attached to the surface.10 Gifford et al.10 demonstrated the detection of target DNA that is labeled by Au NPs using the SPRI technique. In this study, a probe DNA with a hanging sequence template was immobilized on the surface. This hanging sequence is partially complementary to the target DNA. DNA polymerase was used to synthesize DNA sequences on the probe DNA to match the noncomplementary segments of the target DNA. Following removal of target DNA, the surface was exposed to Au NPs functionalized with DNA fully complementary to the surface DNA probe with the synthesized sequences. The SPRI data were collected at different time points and used to calculate the percent reflectivity changes with as little as 0.25% of a monolayer if given enough time to react. This technique can detect 10−100 amol of polymerase product with a partially unknown sequence. On the basis of a similar strategy, the same group detected a surface-bound DNA probe by amplifying the SPRI signal using both Au NPs and in situ transcription of RNA polymerases.14 This method showed excellent sensitivity in the detection of ssDNA down to a concentration of 1 fM in a 25 μL volume. Magnetic NP Enhanced SPR Sensing. Considering the high refractive index and high molecular weight of magnetic nanoparticles (MNPs),20 it is possible to use them as an SPR amplification reagent.8 Wang et al.8 reported the synthesis of monodisperse carboxyl-modified Fe3O4 MNPs for ultrasensitive detection of thrombin using the MNPs as the signal-amplification labels in a sandwich assay with antithrombin molecules immobilized on the Au surface. The resonance angle shifted due to the refractive index change upon binding of the 543



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Figure 2. Schematic representation of different NP LSPR based sensing mechanisms. (A) Au NPs as labels with Ag deposition using hydroquinone (HQ) for signal amplification, (B) LSPR bulk refractive index (RI) sensing, (C) LSPR sensing of local binding events, (D) LSPR aggregation based sensing, and (E) plasmon rulers.

surface. This greatly enhances the optical absorbance/color due to the strong LSPR absorbance band of the Ag. The absorbance can be measured or the assay slides can be examined using a simple flatbed scanner, thus called “scanometric” array. This sandwich based method was reported to greatly enhance the sensitivity and the detection limit via signal amplification. On the basis of this method, Gao et al.33 demonstrated a lectin microarray based resonance light scattering (RLS) assay and screened the binding specificity of 16 different lectins with 4 bacteria and 1 fungus, namely, Escherichia coli, Enterobacter cloacae, Bacillus subtilis, Staphylococcus aureus, and Saccharomyces cerevisiae. First, the primary lectin was immobilized on an epoxide functionalized glass microscope slide in buffer solution. Then, this array was incubated in the microbial strains, followed by the solution with the secondary lectin GS II modified Au NPs. Finally, the microarray was exposed to a 1:1 AgNO3−HQ solution for 6 min for the Ag deposition amplification. These slides were detected with a colorimetric microarray scanner with background subtraction. All five microbes have reasonable binding affinities with GS II, since all of them have terminal GlcNAc residues in their terminal surface glycoconjugates. Depending on their glycosyl recognition motifs on the cellular surface, each bacteria showed a different binding specificity with different lectins. On the basis of these results, the authors built a simple lectin fingerprint for these five microbials. The authors also monitored the lectin binding affinity of the microbial strains in a rich culture medium and a growth inhibitory medium. The binding affinity of the microbial with lectin depended on the growth medium. Screening of serine kinase and tyrosine kinase inhibitors with microarrays using both fluorescence and RLS readout was reported by the same group recently.34 The screening mechanism was as in the previous work; however, they used a dye and Au NP labels as the signal amplification tags. Commercially available dye modified avidin was used in this work. The two readout strategy can improve the accuracy of the assay. The authors demonstrated quantitative determination of the efficiency of both kinase inhibitors.

the properties and applications of colloidal nonspherical noble metal NPs while Li et al.30 reviewed biosensing applications of Au NPs, describing LSPR sensing in one section of the review. Metal NPs As Labels. The simplest form of sensing with metal NPs is to use them as labels, taking advantage of their large extinction coefficient. This allows sensing through standard absorbance measurements, light scattering, or by visual detection. Au and Ag NPs are excellent candidates as labels, since they have a large absorbance band in the visible range and they can easily be functionalized with molecular receptors. A simple strategy is to use a sandwich assay, where a molecular receptor is on a surface, the analyte binds to the surface, and then the receptorfunctionalized metal NPs can also bind to the analyte at the surface. In this way, the metal NP does not attach to the surface unless the analyte is present. The absorbance/scattering or visual observation of the strong metal NP color indicates the amount of analyte present in the solution. This method works well for many analytes, but there have been recent signal amplification strategies that offer very low limits of detection. A signal amplification method was designed to detect DNA and proteins in a metal NP assay based sensing system, using the metal NPs as labels. The first ‘‘scanometric’’ DNA microarray, coupled with a signal enhancement method was based on the attachment of Au NP tags to a surface in the presence of some target DNA, which was followed by electroless deposition of Ag onto the Au NPs for signal amplification. This method was developed by Mirkin and co-workers and offered excellent sensitivity and low detection limits.31,32 In this method, DNA or protein probe receptors were first immobilized on a glass surface. Exposure to target analyte molecules allows them to bind to the surface probes. Then, the surface is exposed to Au NPs functionalized with a secondary antibody or DNA that is partially complementary to the target DNA. In that case the target DNA is partially complementary to the surface probe and partially complementary to the Au NP probe in order to bind the Au NP to the surface. In the end, the Au NP only attaches to the surface if the target analyte is present. The Au NPs catalyze the electroless deposition of Ag onto the glass 544



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Cheng et al.35 reported the ultrasensitive detection of matrix metalloproteinases (MMP) using a similar strategy. In this work, a nitrilotriacetic acid (NTA) modified chip was incubated with Ni2+, which served as the chelating bridge to bind the peptide modified Au NPs. Silver deposition on the chip greatly amplified the signal. In the presence of MMP-7, the peptide-Au NP probes are removed from the surface since the peptide has a MMP-7 specific sequence. In this way, the amount of Au NP probes on the surface depends on the amount of MMP-7 in solution. The quantity of Au NPs on the chip controls the silver deposition and thus the scanometric signal of the sample. The authors demonstrated a detection limit of 0.097 ng/mL for MMP-7 using this method, which was comparable with that of many other techniques. The dynamic range was very broad (0.1 to 100 ng/mL). They extended the application to the detection of MMP-7 in cell culture supernatants, and the results were repeatable, accurate, and stable. Bulk Refractive Index Sensing. Since the LSPR absorbance or scattering wavelength/intensity of a metal NP is related to the refractive index of the surrounding medium, the optical properties of the NP can be used to sense the changes in the refractive index (RI) of the surrounding medium. A red shift of the LSPR band in the spectrum indicates a larger refractive index of the surrounding media. This is called LSPR bulk RI sensing, which can be determined by the LSPR λmax, which is the wavelength of maximum absorbance/scattering.28 The bulk refractive index sensitivity (sλ) is mostly reported in Δλmax per refractive index unit (nm/RIU) or shift of the resonance energy ΔEres per RIU (eV/RIU). The plasmon peak width is not taken into consideration when calculating the bulk RI sensitivity. The peak width is important, though, since the LSPR λmax shift will be easier to detect with a narrow resonance peak as compared to a broader peak. To take this into consideration when describing the overall bulk RI sensitivity, Sherry et al.36 defined and evaluated the figure of merit (FOM) as

Table 1. Summary of Nanoparticles and Their Refractive Index Sensitivities type

λmax (nm)

sensitivity (nm/RIU)

FOM

ref

Au nanoring Au nanostar Au nanodisk Au nanotube Au triangle@SiO2 SiO2@Cu Shell Ag triangle Au NP dimer

ensemble single ensemble ensemble ensemble ensemble ensemble ensemble

1300 725 820 650 1250 610 1093 650

691 218 327 225 737 67.8 1096 471.15

NS 5 NS NS NS NS 4.3 NS

38 39 40 41 42 43 44 45

NS = Not Specified. For example, Dondapati et al.39 studied the sensitivity of a single Au nanostar. They found that a single Au nanostar has multiple LSPR bands using dark-field microscopy. They used Lorentzian peak (multipeak) fitting to the spectrum, which gave four resonance peaks. In their work, the authors exclusively chose nanostars that fit these four resonance bands for further investigation and measured the light scattering spectrum of the single nanostars illuminated with light at different polarization angles (0−180 °C). They attributed the weak and polarization-independent peak around 540−560 nm to the nanostar core. The other three longer wavelength peaks are due to the tips or interaction between the tip and core of the nanostar, which are all polarization dependent. They studied the bulk RI sensitivity of the nanostar using the peak at the longest wavelength (between 650 and 750 nm) in air, water, and different concentrations of glucose solutions. The sensitivity of this specific peak was 218 nm/RIU and the FOM was 5. The detailed shape and structure of metal NPs has a large impact on the refractive index sensitivity. For example, Charles et al.44 evaluated the refractive index sensitivity of 20 different triangular Ag nanoplates with different edge lengths and aspect ratios in solution. With increasing aspect ratio, the λmax of the Ag nanoplates increased in the range of 500−1100 nm. They used different sucrose concentrations in a sucrose−water mixture to vary the bulk RI. The results showed that the LSPR bulk RI sensitivity increased linearly with increasing initial λmax (and aspect ratio) up to a λmax of 800 nm. Beyond 800 nm in the near-infrared (NIR) region, the enhancement was nonlinear. The sensitivity of the NP solution reached a maximum value of 1096 nm/RIU at a λmax of 1093 nm. This is the first detailed study of the dependence of the LSPR sensitivity of solution phase, tunable, plasmonic nanostructures with an LSPR maximum over a large range of wavelengths (500−1100 nm), controlled by their structural parameters. Mirkin and co-workers reported a bulk refractive index sensitivity as large as 737 nm/RIU for silica-protected Au triangular nanoprisms (AuNP/SiO2).42 In their work, AuNP/ SiO2 nanoprisms with three different silica shell thicknesses of 3.2, 13.4, and 22.4 nm were exposed to four different chemical environments, including water, ethanol, dimethyl sulfoxide (DMSO), and tetrahydrofuran (THF). They discovered that nanoparticles with the thinnest shell had the highest refractive index sensitivity. A thin layer of silica coating not only provided high RIU sensitivity but also protected the NPs to increase their stability for sensing applications. Au and Ag are the most common metals used for LSPR sensing because they are fairly stable and have a strong plasmon band in the visible range. Cu has not received much attention,

FOM = [sλ (eV/RIU)]/fwhm (eV) (1) This definition only works for nanostructures with a welldefined resonance peak. With this formula, it is difficult to determine the sensitivity of NPs that do not have a sharp, welldefined resonance peak.37 More recently, Sönnichsen and coworkers37 defined a new FOM* as

FOM* = [(dI /dn)/I ]max = [sλ(dI /dλ)/I ]max

particle

(2)

Here, I is the intensity of the LSPR spectrum, dI/I is the relative intensity change of the resonance spectrum at a fixed wavelength, and n is the refractive index of the surrounding medium. In the same paper, they compared the sensitivity of Au nanorods with different aspect ratio using different measures, including sλ, FOM, FOM*, and the figure of merit for thin layers (FOMlayer*) (another term they defined in the same paper, which we are not discussing in this review). They discovered that, for all definitions of FOM, the optimal aspect ratio for the Au nanorods was 3−4. In recent years, researchers have attempted to find nanostructures with different composition, size, and shape that exhibit high sensitivity.38−45 A summary of different NPs and their RI sensitivities are shown in Table 1. It is well accepted that NPs with sharp edges and tips have higher sensitivity to the environment compared to smoothly curved geometries, such as spherical NPs, because the LSPR field is higher around sharp edges and tips. Motivated by this principle, researchers continue to search for NP shapes that exhibit high RI sensitivity. 545



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substrate compared to the open solution. As a result, the substrate with a larger RI gives a lower sensitivity to the RI changes in solution. To minimize the substrate effect, an isotopic HF etching of the glass substrate was carried out with different etching cycles to create a suspended nanodisk with dielectric pillars. They measured the reflectance of the nanodisk at an excitation angle of 15°. The suspended nanodisk showed a much higher sensitivity of 356 nm/RIU at an etching depth around 28 nm. The sensitivity increased with increasing etching cycles up to three cycles. The authors attribute this to the exposing of the electromagnetic hot spot to the surrounding medium of the nanodisk caused by both exposing the lower part of the nanodisk and redistribution of the electromagnetic field. A further increase in the etching cycles led to significant peak broadening and intensity loss, which the authors attributed to either the removal of the nanodisk from the surface or tilting caused by the unsteady dielectric pillar. The fabrication of NP dimers is another way to enhance the bulk RI sensitivity of metal NPs. The area between coupled NPs exhibits strong electromagnetic field coupling and is considered a “hot spot” for sensing applications. Recently, Cheng et al.45 described the synthesis of conductive polymer coated Au NP dimers (kissing particles) with an interparticle distance of 0−1 nm. First, citrate stabilized Au NPs were encapsulated by a conductive polymer shell and dispersed in methanol. Methanol dissolves the polymer shell, causing aggregation of the NPs, and the polymer forms a monolayer around the aggregated Au NP clusters. The aggregated particles were then dispersed in HCl solution, which leaves the particles either as a dimer or monomer. Further purification removes the monomers. These NP dimers exhibited excellent stability in salt solution up to a concentration of 0.8 M. The Au NP kissing dimers displayed a transverse and longitudinal LSPR band. The bulk RI sensitivity of the dimers was 6 times larger for the longitudinal as compared to the transverse band. Compared to monomers, NP-dimers are much more sensitive for bulk RI sensing. The sensing of smaller molecules is usually more difficult with LSPR, since smaller molecules occupy less volume and lead to smaller changes in the RI.48 However, high-resolution LSPR (HR-LSPR) spectroscopy has made the sensing of small molecules with extremely small wavelength shifts possible through bulk RI sensing of gases.49,50 In a recent study, Van Duyne and co-workers reported the first inert gas sensing and characterization studies based on HR-LSPR spectroscopy.51 The inert gases He, N2, and Ar were used to modulate the environment surrounding Ag and Au NPs fabricated by nanosphere lithography. The gas medium around the NPs was filled with He and then switched to either Ar or N2 at 5 and 10 s intervals. The change in the RI for each switching was only 2.45 × 10−4 and 2.62 × 10−4 for He/Ar and He/N2, respectively. The LSPR Δλmax shift was 0.048 and 0.058 nm. The authors studied the shift in the LSPR spectrum with time, which indicates that the introduction of gases only changes the bulk RI and does not bind or adsorb directly onto the NPs. Van Duyne and co-workers reported the sensing of CO2 and SF6 over Ag NPs using HR-LSPR spectroscopy.48 They used an array of 40 nm diameter Ag NPs fabricated by nanosphere lithography. The sample was purged with dry N2 overnight before use and then CO2 or SF6 was introduced into the surrounding medium of the NPs. The target gas was switched on for 60 s and then switched to N2 for another 60 s. The cycle was repeated five times. A significant red shift of the LSPR peak (0.17 nm for SF6 and 0.13 nm for CO2) was observed upon

since Cu easily forms Cu oxide, which affects the optical properties. However, Cu can be used for LSPR sensing, since it possesses similar optical properties as Au and Ag and is relatively cheaper. Kim et al.43 reported a procedure to synthesize silica/Cu core/shell NP arrays on an Au film. The authors termed this a multispot copper-capped NP array (MC-NPA) chip. First, the glass substrate was coated with a 40 nm Au film. Next, they placed a silica-rubber mask onto the surface to control the fabrication of the multispot NP attachment. A self-assembled monolayer (SAM) of 4,4′-dithiodibutyric acid was formed by soaking the glass−Au film in the solution for 1 h. They modified the surface of the silica NPs with (3-amino-propyl)triethoxysilane and used 1-ethyl-3(3-dimethylaminopropyl)carbodiimide to attach onto the Au surface. Finally, the Cu cap was deposited onto the silica NPs using electron beam evaporation. Since copper oxides tend to form on the Cu, even at room temperature, and the layer of copper oxide affects the optical properties of the Cu, the sample was soaked in an acetic acid solution for 20 s before use every time to remove the copper oxide. The authors found that the optical property of the MC-NPA depends on the number of Cu layers deposited. Each layer of Cu was 10 nm. The authors monitored the extinction spectrum of the MC-NPA after each layer up to a thickness of 100 nm. The extinction intensity increased up to three layers (30 nm) but then decreased with increasing peak broadening as the thickness of the Cu shell increased. The authors attributed this to an insufficient Cu coating ( dT30 > dT70, due to the fact that SSB binding to shorter DNA is highly cooperative compared to the longer DNA.64 This group also studied binding of SSB to dT20, dA20, dC20, and dG20, showing that SSB binds to dT20 and dA20 more effectively. In the same study, the authors detected sequence specific DNA hybridization. DNA strands were preincubated with the SSB to form SSB-ssDNA, which helps to stabilize the Au NPs against aggregation. Introduction of cDNA target will hybridize with the probe ssDNA, which will cause aggregation of the Au NPs since there is no ssDNA to form the SSB-ssDNA stabilizing complexes. In contrast, mismatched DNA cannot hybridize with the probe DNA, which leaves the SSB-DNA complex intact to prevent aggregation of the NPs. This strategy can detect a single base pair mismatch, even if it is located at the extreme end of the DNA. Proteins. It is well-known that there are highly specific interactions between metal ions and specific amino acids, so that bioconjugated NPs can cross-link to aggregate in the presence of specific metal ions, which can cause a shift in the LSPR plasmon band of the NPs. On the other hand, if the NPs can be modified by the metal ion, then they can be used to detect protein with specific amino acids based on the aggregation of the NPs. For example, on the basis of the strong coordination between myoglobin (Mb) and Cu2+, Du and coworkers demonstrated the qualitative and quantitative detection of Mb by iminodiacetic acid (IDA)-functionalized Au NPs.65 They prepared the Au NPs via a seed mediated growth procedure, functionalized with IDA, and then treated with Cu2+ before exposure to Mb. No color change occurred upon addition of Cu2+, but the color changed from red to purple after addition of Mb with a corresponding red shift from 518 to 552 nm due to aggregation of the NPs caused by cross-linking of IDA-Cu2+-histidine (His) residues of Mb. The LSPR λmax red shift was linear over the concentration range of 0.91− 18.2 μg/mL of Mb. The authors also used this strategy for sensing pepsin, HSA, BSA, and BHb. Pepsin and HSA did not show any color change because these proteins either do not have any His residue or do not have any surface His residues. BSA has two His residues, but only one can coordinate with Cu2+; no cross-linking aggregation and therefore no color change occurred. BHb showed some color change and a much smaller LSPR shift compared to Mb. The authors attribute this to steric hindrance by the larger BHb molecules. These results indicate that the sensor response is related to the number and distribution of surface His residues as well as the size of the protein. Metal Ions. Noble metal NP aggregation was applied for the colorimetric detection of heavy metal ions since there are distinct color changes easily discernible by the naked eye. Ling and co-workers demonstrated the detection of Ag+ with Au NPs by LSPR light scattering spectroscopy.66 Citrate stabilized 13 nm diameter Au NPs were mixed with oligonucleotides (oligo-1 or oligo-2). Oligo-1 and oligo-2 are ssDNA that contained the same nucleic acid sequence with different order. Oligo-1 had a specific sequence that contained five CytosineCytosine (C−C) mismatched base pairs. Therefore, oligo-1 can self-hybridize in the presence of Ag+ via C−Ag+-C base pairs. Oligo-2 served as the control. First, the authors mixed the Au NPs with NaNO3 and the oligonucleotide solutions. The presence of salt should cause salt induced aggregation of the NPs; however, no color changes were observed for this mixture due to the strong adsorption of the ssDNA on the NPs. The 550



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Small Molecules. Functionalized NPs can aggregate in the presence of small molecules through hydrogen-bond or electrostatic interactions, acting as a label-free detection method. For example, Huang and co-workers detected melamine using polythymine-stabilized Au NPs.71 The triple H-bonds between the melamine and thymine caused the aggregation of welldispersed polythymine stabilized Au NPs. The existence of the triple H-bonds were confirmed by NMR. Upon aggregation, the color of the solution changed from red to blue via purple as confirmed by LSPR absorption and scattering spectra. The intensity ratio between the new band at 620 nm and original 520 nm was calculated versus the concentration of the melamine as well as the intensity increase of the scattering band at 560 nm. Both showed a linearity range from 80 to 1000 nM, with a detection limit of 20 nM. The authors also discovered that Au NPs stabilized with oligomers of other compositions (like polyAn, polyCn, and polyGn) cannot aggregate in the presence of melamine, since they cannot form the triple H-bonds with melamine. The degree of aggregation of the NPs increased with increasing chain length for polythymine. This study represents the simple, fast, label-free detection of melamine. Since the authors did not study the specificity, the detection of melamine in real samples has to be evaluated. On the basis of electrostatic interactions between streptomycin and mercaptoacetic acid (MPA) stabilized Au NPs, a colorimetric detection of streptomycin in raw milk was reported.72 Streptomycin is an antibiotic drug, with a structure of two guanidyl groups, which have a pKa around 13.5. This high pKa causes the streptomycin to bear positive charges in aqueous solution. Electrostatic attraction between these two positive charges and the negatively charged group on the surface of the MPA stabilized Au NPs caused the cross-linking aggregation of the Au NPs. The color of the solution changed from red to blue, which indicated the presence of streptomycin. The intensity ratio between absorption bands at 700 and 520 nm were monitored as a function of the streptomycin concentration. A700/A520 increased slightly when the concentration of streptomycin was below 0.6 μM and increased dramatically from 0.8 to 1.8 μM. An optimized sensing strategy was designed in which 0.6 μM streptomycin was added into the solution before sensing. This optimized probe showed a detection limit of 2 nM. The selectivity was evaluated in the presence of antibiotics, molecules with positively charged groups, metal ions, other biomolecules, and some stabilizing agents for Au NPs. All of these molecule or ions did not interfere with the detection of streptomycin. Under optimal conditions, the addition of 50 ppb streptomycin led to a distinct color change from red to purple in raw milk samples. Plasmonic Rulers. The overlapping of the electromagnetic field of two metal NPs in close proximity causes an LSPR peak shift according to the interparticle distance of the NP dimer. A red shift and an intensity increase is usually observed when the interparticle distance of the dimer decreases. This phenomenon can be utilized to measure the distance between the NPs in the dimer. This strategy has been termed “plasmon ruler”. Chen et al.73 described the sensing of DNA based on an increase in distance of Au NP dimers that were linked together through a hairpin DNA loop (plasmon rulers). The λmax of the scattering of the light blue-shifted 11 nm upon addition of cDNA (cDNA), since the hybridization of the DNA caused an increase in the interparticle distance of the dimers. They monitored the Δλ/λ as a function of the target DNA concentration

as low as 0.1 pM in the buffer solution. The signal can be differentiated from nonspecific binding of non-cDNA at that low concentration. They further evaluated the detection of target DNA in serum at a concentration up to 50%. At 75% serum, the nonspecific binding of proteins dominated. However, the degree of the plasmon band shift allowed differentiation between nonspecific and specific binding. One of the challenges of plasmon rulers is calibration of the ruler. Since the LSPR of the NPs depends on many factors including the size and shape of the NPs, a slight difference in the size or shape of the NP causes a variation of the LSPR of the dimer. This variation of the LSPR of the dimer led to an uncertainty of the measurement of the interparticle distances of a new NP dimer.74 The Reinhard group reported the calibration of a DNA-tethered silver plasmon ruler.75 In this study, they used commercially available Ag NPs with an average diameter of 41.0 nm. The Ag NPs were first functionalized with ssDNA and then mixed to hybridize and form dimers. The dimers were separated from monomers via gel electrophoresis and recovered through gel electroelution. The dimers were finally immobilized on a TEM grid. Only dimers of the spherelike NPs were selected for further study. The plasmon resonance energy (Eres) of the dimer was directly correlated with the ratio of the center-to center particle separation (L) to diameter (D) of that exact dimer. A continuous red shift of the Eres was observed with decreasing L/D for dimer that has an interparticle distance between 1 and 25 nm. The calibration curve showed that the resonance energy decreased exponentially with an increase in interparticle distance. When the interparticle distance is smaller than 1 nm, the Eres did not continue to decrease. The authors attributed this to the direct charge transfer of the nearly touching particles. Liu et al.76 reported the first three-dimensional plasmon ruler based on coupled Au nanorods. Using high-precision electron beam lithography and layer-by-layer stacking nanotechnology, five Au nanorods with different length and orientation were stacked to form the 3D plasmon ruler. The structure of the 3D plasmon ruler is shown in Figure 4A. The five nanorods were stacked as the letter H, in which the red rod was stacked in between the parallel green rod pair and parallel yellow rod pair. The whole system was placed on a glass surface in a homogeneous dielectric medium. This particular nanorod stacking structure was chosen since the two parallel nanorod pairs gave two quadruple resonances and those two resonances coupled to the middle nanorod for a dipole resonance. The authors found that only the complete asymmetry of the system can cause distinct and sharp resonance peaks. As shown in the top spectrum of Figure 4B, the shift of the sharp transmittance peaks I and II were monitored upon the moving of the middle red rod laterally (displacement of S) or vertically (displacement of H). The transmission intensities of both resonance I and II decreased with the decreasing of the S, since this decreased the asymmetry of the system. The higher energy resonance II decreased faster than the lower energy one because the resonance I represents dipole quadruple coupling of the top long nanorod pair and resonance II represents that of the bottom short pair. The displacement of S has a larger affect on the quadruple resonance of the shorter rods. The vertical increase of H caused the increase of the transmission intensity of resonance I and decrease of that of resonance II (The displacement toward the upper rod pair was defined as +H the other way was −H). The authors attributed this to the reduced coupling due to the 551



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Figure 4. (A) Schematic diagram of the 3D plasmon ruler. (Inset) Definitions of the geometrical parameters. The red rod is displaced from the symmetry axis of the bottom green rod pair by S. The lengths of the top yellow and bottom green rods are L1 and L2, respectively. The vertical distance between the red rod and the yellow rod pair is H1, and that between the red rod and the green rod pair is H2. E, electric field; H, magnetic field; k, direction of light propagation. (B) Calculated transmittance spectra of the 3D plasmon ruler. Bottom curves: S = 0 nm, L1 = 310 nm, and L2 =310 nm (ΔL = L1 − L2 = 0 nm). Middle curves: S = 40 nm, L1 = 310 nm, and L2 = 300 nm (ΔL = 10 nm). Top curves: S = 40 nm, L1 = 340 nm, and L2 = 270 nm (ΔL = 70 nm). I and II transmittance peaks develop inside the broad dipolar absorption dip, depending on the degree of structural symmetry breaking. Reprinted with permission from ref 76. Copyright 2011 AAAS.

Aggregated Nanoparticles. Wu et al.81 demonstrated the imaging of Cr3+ in living cells using citrate-capped Ag NPs. Cr3+ caused the aggregation of the citrate-stabilized Ag NPs. Cr3+induced aggregation is highly specific in the presence of many other cations, including K+, Na+, Ca2+, and Mg2+. The authors attributed this high specificity to the high specific chelation of Cr3+ with citrate ions. The aggregation caused the solution of the Ag NPs to change color from yellow to pink with a decreasing intensity of the absorption band at 392 nm and appearance of a new band at 560 nm. The light scattering of the NPs was monitored during this procedure. The original scattering band at 452 nm blue-shifted upon the addition of Cr3+ and a new band appeared at 560 nm. Both the absorbance ratio A560 nm/A396 nm and the intensity of the new scattering band I560 nm were plotted versus the concentration of Cr3+. The linear range was from 0.5 to 10.0 μM, and the limit of detection was 0.13 and 0.08 μM, respectively. The authors demonstrated the imaging of Cr3+ in vitro and in vivo with citrate-coated Ag NPs using dark-field microscopy. Cells preincubated with Cr3+ showed a significant increase in LSPR scattering intensity at 560 nm, while native cells did not. Fluorescence Spectroscopy. Introduction. Fluorescence (FL) is one of the most popular methods of detection in analytical chemistry because of its high sensitivity. There are still some well-known limitations. Most conventional fluorophores suffer from photobleaching, short lifetime, and limited brightness.82 NPs of various materials have shown unique optical properties due to their large surface to volume ratio and unique electronic structure that has aided in the FL detection of various analytes. Compared to organic dyes, fluorescent NPs are often more stable and resistant to photobleaching and have higher luminescent intensity and longer lifetime. Most importantly, their emission is narrow and symmetrical and is tunable by size, shape, and composition.83 These features broaden the application of NPs toward FL sensing and imaging, especially for biomolecules and heavy metal ions. This section of the review summarizes recent FL-based sensing applications of NPs. There are different sensing strategies, including “turn-on” sensing, “turn-off” sensing, and ratiometric sensing. “Turn-on” sensing occurs when the FL is initially quenched usually through the fluorescent resonance energy

farther distance between the plasmon of the middle nanorod and either the upper or lower nanorod pairs. Creation of Metal NPs. Wang et al.77 demonstrated the detection of hydroquinone based on the LSPR-light scattering (LS) of Ag NPs that were created by Ag+ reduction by hydroquinone in a solution of NaOH and ammonia. The color of the solution changed from colorless to slightly yellowish color, which gives an absorbance peak at 430 nm. TEM confirmed the generation of Ag NPs. In the NaOH concentration range of 0−2.5 × 10−4 M, the intensity of the scattering spectrum increased and then decreased, being highest at a concentration of 1.5 × 10−4 M. An ammonia concentration of 3 × 10−3 M was optimal. The intensity of the LSPR-LS at 500 nm increased linearly in the range of 0.4−2.5 μM of hydroquinone with a 70.6 nM detection limit. LSPR Imaging. Distributed Nanoparticles. Noble metal NPs are one of the promising tools for optical bioimaging due to the strong light scattering at the LSPR frequency, typically in the visible range.78,79 The scattering light intensity is sensitive to the size, shape, and interparticle distance of the NPs.79 Typically, NPs are labeled with specific receptors that interact with molecules in biological samples. Huang and co-workers described intracellular protein imaging using aptamer-functionalized Ag NPs, where the aptamer served as the specific recognition agent and Ag NPs as the illuminophore.80 Streptavidin-coated Ag NPs were functionalized with aptamer (Apt)-labeled biotin to generate the Apt-Ag NP probes. This nanoprobe was used to image the intracellular endocytic pathways of prion protein (PrPc) in human bone marrow neuroblastoma (SK-N-SH) cells. Dark-field light scattering images showed that the NPs were monodisperse in the cell but exhibited multicolors. The authors attributed these different colors to the size and shape of different NPs as well as the different refractive indices of the surrounding medium. By labeling the PrPc with the Apt-Ag NP probes, the authors studied the intracellular pathway of PrPc. TEM images revealed Ag NPs in the cytoplasm but not in the nucleus. They were distributed in the plasma membrane, endocytic structure, lysosome, and mitochondria. This indicated that Apt-Ag NP labeled PrPc internalized into SK-N-SH cells via a caveolae-related endocytic pathway. 552



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QDs were first incubated in a Na2S solution while monitoring the FL intensity. The coating of S2− quenched the FL of the QDs. Addition of Zn2+ or Cd2+ resulted in recovery of the FL signals by forming a ZnS or CdS shell around the QDs. The authors demonstrated the selective detection of Zn2+ and Cd2+ over other metal ions in real water samples. Unfortunately, this method is not suitable for intracellular imaging because of Cd2+ toxicitiy. QDs are also excellent substitutes over traditional FRET based dyes because of their excellent photophysical properties as mentioned in a previous section. The broad absorption spectrum means that different colors of QDs can be excited by a single excitation source as compared to traditional dye, which has to be excited within a narrow absorption band. This property together with the narrow symmetrical emission band makes QDs ideal for multiplexed optical sensing, especially for biological and medical applications. With multiplex sensing, several parameters can be determined simultaneously with a single measurement, significantly decreasing the time and cost associated with the measurement. The conjugation of QDs with highly ion-specific DNAzymes as a FRET sensor was recently demonstrated for multiplexed detection of Cu2+ and Pb2+.92 In this paper, the authors used silica-modified CdSe/ZnS core/shell QDs. The silica layer prevented nonspecific binding of heavy metal ions to the QD, which can quench the FL. DNAzymes were attached to the QD through a zero length cross-linker, and both substrate and DNAzymes were labeled with a quencher. In the absence of the target metal ion, the quencher on the DNAzymes is in close proximity to the QD. Binding of the DNAzymes with the target metal ion causes the substrate to cleave off from the QD, restoring the FL of the QD. The authors found that the FL intensity increased linearly for Cu2+ and Pb2+ in the concentration range of 1−50 nM, with a detection limit of 0.5 and 0.2 nM, respectively. They also demonstrated multiplexed sensing of Cu2+ and Pb2+, since they have different emission wavelengths of 625 and 530 nm, respectively. QDs can also serve as the acceptor in the FRET process. Hildebrandt and co-workers93 demonstrated a 5-fold multiplexing QD biosensor in which it served as the FRET acceptor. Luminescent terbium complexes (LTCs) were used as the FRET donors in this example because of their long excited-state lifetimes (up to milliseconds), large Förster Radii (up to 11 nm), and well separated emission bands with the QDs. LTCs were bound to QDs through biotin−streptavidin interactions. Five different commercially available QDs were used as energy acceptors, with emission maxima at 529, 565, 604, 653, and 712 nm. Mixing the biotin labeled QDs with the streptavidin labeled LTCs led to the FRET process, resulting in quenching of the LTCs but increased emission of the QDs. The QD emission was monitored with increasing concentration of the QDs. The results showed low spectroscopic cross-talk between the five different QDs, and the technique was fast, simple, and sensitive. Dong et al.94 reported the first FRET based biosensing using a QD-graphene oxide (GO) donor−acceptor pair. In this method, the QD was the fluorophore used to label a molecular beacon (MB), a hairpin shaped oligonucleotide probe that can report the presence of specific target nucleic acids. Compared to the ssDNA, MB has much higher specificity. The π−π stacking interaction and hydrogen bonding between the −OH or −COOH groups of GO and −OH or −NH2 groups of the MB brought the QD into close proximity to the GO (3.24 nm),

transfer (FRET) process but then turned on through interaction with the analyte, which separates the fluorophore and the quenching agent. Fluorescent resonance energy transfer (or Förster resonance energy transfer, FRET) is a process involving the distance dependent energy transfer from a donor molecule to an acceptor molecule through dipole−dipole interactions.84 The energy transfer rate is inversely proportional to R6, where R is the distance between the donor and acceptor. FRET is the basis of many sensing platforms where the recognition of an analyte molecule of interest brings the donor and acceptor into close proximity for detection by the FRET process. Usually fluorescent NPs serve as the FRET donor and a molecular dye serves as the acceptor. The close proximity of the acceptor to the NPs decreases the emission intensity of the NPs and increases the acceptor emission intensity.85 In “turn-off” sensors, the FL of the NPs usually becomes quenched by some acceptor molecule or surface (metal or carbon), which occurs by the NP coming in close proximity to the acceptor or quenching surface through binding with target analyte. The intensity loss in FL is proportional to the concentration of analyte. In ratiometric sensing, there is usually a reference dye in the sensing system, which serves as the internal standard since the FL intensity of it is not affected by interaction with the target molecule. The FL intensity ratio of the NP and the reference was measured as the signal. Ratiometric measurements can overcome interference from the incident light, the absolute concentration of the fluorophore, and losses due to photobleaching. The types of NPs described in this review include quantum dots (QD), metal NPs, rare earth doped NPs (up-converting NPs), dye-doped silica NPs, carbon NPs, and polymeric NPs. We also briefly review a few recent examples of NP biobarcodes which can lead to multiplexed identifications of multiple targets. There are some excellent recent reviews describing the properties and applications of NPs in FL detection. Zhong published a comprehensive review of nanomaterials in FLbased biosensing up to 2008.86 Baù et al.82 recently reviewed chemosensors with fluorescent NPs, mainly QD and dye-doped particles. There are also reviews on specific fields, such as sensing and imaging in genomics and proteomics87 and bioimaging and drug delivery,88 for example. “Turn On” Fluorescent Sensors. Quantum Dots. QDs are luminescent semiconductor nanocrystals with diameters of 2− 10 nm, composed of elements such as Cd, Zn, Se, Te, In, P, and/or As. It has been known for some time that QDs are fluorescent and that the emission wavelength is size tunable; it decreases with decreasing particle size due to an increase in the semiconductor band gap with decreasing size. QDs possess excellent photophysical properties and high quantum efficiency, inspiring researchers to broaden their application into many different areas, such as biological systems. The first biological application of QDs was reported in 1998 by Bruchez et al.89 and Chan et al.90 The intrinsic FL of QDs made it possible in sensing applications by directly measuring the change in the FL intensity of the QDs due to their interaction with analytes. There are emerging applications of QDs that operate in a “turn-on” mode for heavy metal ions, which can decrease the chances of false positives compared to the “turn-off” mode, since many things can lead to FL quenching of QDs. Xu et al.91 reported the detection of Zn2+ and Cd2+ in an aqueous solution using S2− modified CdTe QDs as a “turn-on” FL probe. Water-soluble 3-mercaptopropionic acid-capped CdTe 553



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QDs not only have an intrinsic FL signal themselves but also the ability to enhance the FL signal of nearby fluorophores. There are some sensing and imaging applications based on QDamplified FL. The Cd2+ ions that are dissociated from a QD trigger the FL emission of nonfluorescent dye, such as Fluo-4 and Rhod-5N upon binding.97 On the basis of this fact, Han et al.98 designed a signal amplification of the FL of Rhod-5N for the in situ monitoring of carbohydrates in living cells. First, CdS QDs were labeled with 3-aminophenylboroic acid (APBA), which can recognize and bind to the sialic acid (SA) groups on a cell surface. The authors also introduced glyconanoparticles (sialic acid modified Au NPs) into the system, which can cause the multiplex sandwich binding of the QD to the cell surface for dual signal amplification purposes. Those samples were treated with HNO3 to dissolve the QDs. After adjusting the pH to 7.4, Rhod-5N was introduced and the triggered FL was monitored. The FL intensity was proportional to the logarithmic value of the cell concentration in the range of 500 to 1.0 × 107 cells/mL, with a limit of detection of 20 cells/mL. Duan and co-workers99 described the use of phenylboronic acid (PBA) conjugated CdSe/CdS/ZnS core/shell/shell QDs for labeling sialic acid (SA) on living cells. These QDs were coated with an amphiphilic copolymer of maleic anhydride and octadecene (PMO), which was reported to prevent leaking of cytotoxic Cd2+.100 SA is found in glycoproteins and are at higher levels in colorectal cancer cells compared to normal cells.101 PBA can recognize and bind to SA on the cell surface. After binding, the cell membranes were stained for 1 min and monitored by confocal microscopy. There was a much lower FL intensity from the cell surface when they were placed in a solution containing free SA, which competitively binds the PBA. The authors studied the diffusion of sialylated glycoprotein by tracking the FL signal of the functionalized QDs. This labeling method is simple, is fast, and does not require pretreatment of the cell. Metal Nanoparticles. It is well-known that metal NPs can quench the fluorescence of fluorophores, but in recent years researchers have found that small metal NPs (or clusters) exhibit fluorescence. Accordingly, these NPs have been employed in sensing and imaging applications. Lan et al. used fluorescent water-soluble DNA-Ag nanoclusters (NCs) for Cu2+ ion detection.102 A FL enhancement occurred for these probes in the presence of Cu2+, making them highly sensitive with a 8 nM detection limit. FL occurred from DNA-Ag NCs due to their lower density of electronic states. In the presence of Cu2+ ions, the DNA becomes more rigid and as a result, the DNA protects the Cu2+−DNA−Ag NCs more efficiently from environmental quenching. These are highly selective to Cu2+ ions compared to other metal ions ions by a factor of 350-fold. The effect of pH on Cu2+ detection was relatively small but optimal at pH 6.0. Cu(OH)2 formed at higher pH, which resulted in less DNA-Cu/Ag NCs and lower FL enhancement. A decrease in the negative charge density on DNA at lower pH also hindered Cu2+ ion binding to the NC surface, which resulted in lower FL. Reaction times were shorter at 60° and 80 °C but DNA-Cu/Ag NCs were also unstable and a lower fluorescence signal appeared at these temperatures due to the uncontrolled reduction of Cu2+ ions and increased collisions with NCs. At temperatures higher than 80 °C, the folded structure of DNA is less stable, which results in the decreased capping ability of DNA. The optimal reaction temperature and time was 40 °C and 30 min, respectively. The authors determined the Cu2+ ion content in pond water and

causing energy transfer from the QD to GO and quenching efficiency up to 90%. A schematic representation of this sensor is shown in Figure 5. Hybridization of the cDNA strand with the MBs on the QD increases the distance between the QD and GO out of Förster radius, so that there is no FRET, restoring

Figure 5. Schematic illustration of graphene oxide (GO)-induced FL quenching of MB-QDs coated with hairpin DNA and the FL enhancement that occurs when the hairpin opens in the presence of analyte. Reprinted from ref 94. Copyright 2010 American Chemical Society.

the FL intensity of the QD up to 64.7%. They tested three different DNA strands using this sensor: cDNA, single base pair mismatch, and three base pair mismatch. The cDNA strand showed 2.5 times more relative FL intensity compared to the single-based mismatch, indicating good selectivity. Using the same idea, the authors tested the response to a different concentration of thrombin using a thrombin binding aptamerlabeled QD. The method has good potential for the detection of the protein; however, FL self-quenching by thrombin occurred when above 200 nM, which the authors attribute to aggregation of the aptamer-QD. Since the FRET process depends on the distance between the donor−acceptor pair, it can follow biocatalytic actions, such as enzymatic cleavage of biomolecules. Willner and co-workers95 published an excellent perspective on sensing applications by this mechanism. Liang and co-workers96 reported the first QDFRET based multiplexed sensor for the detection of endonuclease activity. Two different kinds of QDs were used with FL maxima at 525 and 605 nm. QD525 and QD605 were attached with 4-[4-(dimethyl-amino)phenylazo]benzoic acid N-succinimidyl ester (DABCYL)-labeled DNA and Black Hole Quencher-3 succinimide ester (BHQ-3)-labeled DNA strands, respectively. Then, the quencher labeled-cDNA strands were hybridized to the QD conjugated DNA strands to form the QD-based nanoprobes. There was no FL of QDs due to the close proximity of the quenchers at first. After adding the target endonucleases EcoRI and BamHI, the DNA strands were cleaved and disrupted, which caused the quenchers to leave the QDs, restoring the FL. The restored FL intensity depends on the concentration of each target endonuclease. The FLs of both QDs were monitored at the same time without any cross-talk of their FL peaks. The authors also evaluated the activity of endonuclease inhibitors. They showed that this is a simple, specific, and sensitive technique for multiplexed detection of activities of both endonucleases and their inhibitors. 554



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experiences a conformational change to form a more compact structure, which provides better protection to the Ag NCs from its surrounding environment that is responsible for FL quenching. dC12-AgNCs enabled the detection of glutathione, Cys, and Hcy at a limit of detection of 6.2, 2.1, and 1.5 nM, respectively, which is much lower than the concentration of these thiols in plasma. The response time is less than a minute, the maximum FL enhancement occurred at pH 7.0 in the presence of thiols, and the optimal temperature for the assay was found to be 25 °C as DNA was unstable at higher temperatures. Compared to other amino acids and biologically relevant analytes, dC12-AgNCs probes could specifically detect glutathione, Cys, and Hcy and the FL enhancement increased with increasing charge donating capability of the thiol compounds. Thiol compounds in human plasma were detected using dC12-AgNCs. Werner and co-workers used DNA templated Ag nanoclusters (DNA/Ag NCs) for target DNA detection.106 In this example, the authors utilize DNA with a nucleation sequence and hybridization sequence. The Ag NCs form on the nucleation sequence of the DNA and exhibit a very week green FL emission. Hybridization of a complementary target (containing an overhanging guanine rich tail) on the hybridization sequence leads to a strong red fluorescence emission with an enhancement ratio as large as 500. The authors showed that the red FL emission of the Ag NCs occurred as a result of the guanine bases being in close proximity to the Ag NCs. The FL emission increased exponentially with an increasing number of guanine bases close to the NCs, which was controlled by altering the overhanging guanine rich tail. Since a true target DNA would not necessarily have a guanine rich tail, the authors designed a nanocluster beacon (NCB) which has a separate Ag NC probe and guanine rich probe which are brought together through hybridization with target DNA, leading to guanine bases near the Ag NC and red emission. This led to a 76-fold increase in red emission as compared to the signal in the presence of nonspecific target or no target. They showed a signal-to-background (S/B) ratio of 175 for the detection of an influenza target as compared to 32 for molecular beacons. Jiang and co-workers fabricated rhodamine B coated Au NP arrays on an elastomeric substrate of poly(dimethyl-siloxane) (PDMS) and monitored the FL intensity change as a function of pressure.107 With an increase in pressure, the elastic substrate became compressed and the distance between the Au NPs decreased, resulting in an increase in the FL intensity. The FL intensity decreased after relief of the pressure. Rare-Earth Nanoparticles (Up-Converting Nanoparticles). Rare-earth based NPs (RENPs) usually consists of a host particle matrix doped with rare-earth light emitting ions.108 NaYF4 is a common host matrix for sensing due to its low photon energy. Lanthanum, ytterbium, erbium, and thulium ions (itself or codoped) are the most popular rare-earth ions that are doped in the host matrix. These NPs show unique and interesting optical properties, making them one of the hottest topics for research in biological sensing and imaging. They exhibit no photobleaching or blinking, no size-dependent optical properties, large antistokes shifts (up to several hundreds of nanometers), low cytotoxicity, long lifetimes, and narrow emission and absorption bands. One of the most important optical properties is that multiphoton excitation of NIR light can lead to emission at higher energy in the UV−vis-NIR range, termed up-converting phosphor (UCP).108,109 In biological applications, the NIR light can penetrate into the tissue deeper, and will not excite other fluorophores inside the tissue, which

Montana soil to demonstrate their use in real environmental samples. Borghs and co-workers reported theoretical and experimental studies of FL quenching and enhancement near Au NPs using a hairpin DNA probe of various lengths labeled with a fluorophore and various sized Au NPs.103 When the hairpin probe is on the surface of the Au NP, the fluorophore is in close contact with the Au NP, resulting in nearly perfect quenching of FL for all DNA hairpin lengths and Au NP sizes (20− 100 nm diameter). Upon hybridization with target DNA, the hairpin opens and stretches out, resulting in the fluorophore moving well away from the Au NP where it is no longer fully quenched. The authors showed that the FL increased for all Au NP sizes and hairpin lengths after target hybridization, but that the FL was lower than 100% of the FL for the free fluorophore in solution, indicating some quenching even at long distances from the NP. In contrast, the 100 nm diameter Au NP exhibited enhanced FL for the fluorophore at longer distances relative to the free flouorophore in solution (123%). Since light absorption is larger than scattering for the smaller particles, some quenching occurs at all of the fluorophore−Au NP distances studied. In contrast, scattering dominates for larger NPs, allowing enhancement of FL with the fluorophore at the larger distances from the Au NP. On the basis of the fundamental studies, the authors developed a DNA sensor by using the 100 nm diameter Au NPs with a 35 base pair DNA and reported a detection limit of 100 pM of target DNA. Cu ions were detected by recovering the FL of DNA-Cu/Ag NCs that were quenched by mercaptopropionic acid (MPA).104 The Ag NC capping ability of DNA was reduced in the presence of thiols since the thiols weaken the interaction between DNA and the metal cluster, resulting in FL quenching. In the presence of Cu2+, the thiol-induced FL quenching is lower due to formation of a Cu(SR)2 complex, which is further oxidized to a dithiol. The quenching efficiency of the thiol depends on the formation constant for Cu and Ag−thiol complexes, and the rate of thiol oxidation increased with an increase in basicity. Cu2+-induced FL enhancement was largest for solutions with MPA compared to 2-mercaptoethanol and cysteine and optimal at pH 8.0. Cu2+ induced recovery of FL was relatively lower at pH 6.0 due to the weaker interactions between the more protonated MPA and Cu/Ag NCs. At pH 10, Cu2+ reactivity decreased due to the formation of Cu oxides and hydroxides. These probes were selective to Cu2+ over other metal ions by a factor of 2300 and detected Cu2+ at 2.7 nM. Cu2+ ion content in soil and pond water samples was determined to demonstrate environmental applications of these probes. The fluorescent properties of NCs depend on the nature of the template used to synthesize the NCs.105 When DNA is used as a template, the effect is even more prominent.105 In this regard, Qu and co-workers developed a FL “turn on” assay for thiol detection by modulating DNA-templated Ag NCs.105 For the same thiol molecules with different DNA (different length and base composition) the authors observed different response patterns, including, no change in fluoresecence, a decrease in FL intensity, and increase in FL intensity. AgNCs formed with different DNA templates displayed different FL response patterns in the presence of thiols. FL enhancement occurred in the presence of thiols for dC12 DNA Ag NCs. The authors attributed the enhancement to charge transfer from the ligand to the metal center upon the formation of an Ag−S bond and a change in the DNA microenvironment caused by the addition of thiols. In the presence of thiol, the DNA 555



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can lead to higher resolution. Haase and Schäfer110 published a comprehensive review about up-converting NPs recently. Typically, in a FRET process, rare-earth based NPs serve as the donor and a conventional fluorophore as the acceptor. The advantage of using RE NPs as the donor is that the NIR excitation source will not excite the acceptor, giving higher sensitivity. FRET processes involving UC-NPs have also been used for sensing biomolecules. Peng et al.111 demonstrated sensing of glucose using a bioconjugated UC-NP and Au NP donor− acceptor pair. UC-NPs were labeled by Concanavalin A (Con A) and Au NPs by thiolated β-cyclodextrins (SH-βCD). Con A can specifically bind to glucose or mannose. The sensing mechanism is based on the competitive binding of Con A to free glucose in the solution or the glucopyrasosyl subunit of dextran on the Au NPs. The binding of Con A to dextran brings the Au NPs into close proximity with UC-NPs, enabling the FRET process. In the presence of glucose, the UC-NPs detach from the Au NPs, suppressing the FRET process. The FL intensity of the UC-NPs (illuminated at 980 nm) increased with increasing glucose concentration in the range of 0.4 − 10 μM with a detection limit of 0.043 μM. The authors also demonstrated the detection of glucose in pretreated human serum and real human serum, which showed the same linear range and a detection limit of 0.065 μM. Liu et al. demonstrated the application of these NPs for bioimaging of cyanide.112 In this study, they coated the NP with chromophoric iridium(III). Iridium showed a strong broad absorption band at 505 nm. Spectral overlap with one of the absorption band of the NPs leads to FRET. Interaction with the cyanide ion resulted in suppression of the absorption band at 505 nm and suppressed the FRET process. The existence of α,β-unsaturated carbonyl groups on the iridium led to strong interactions with cyanide, thereby disrupting the π-conjugation system.113 The addition of cyanide into the iridium coated NP complex led to recovery of the green emission of the NPs, whose intensity increased linearly with cyanide concentration in the range of 0−10 equivalences until reaching saturation at 15 equivalences of cyanide. The detection scheme showed high selectivity toward cyanide and they imaged the cyanide in HeLa cells. Dye-Doped Silica Nanoparticles. Dye-doped silica nanoparticles (DDSNs) consist of silica matrix NPs doped with a large number of dye molecules inside.114 In 1968, Stöber developed the first controlled preparation of monodispersed colloidal silica spheres.115 Compared to QDs, silica NPs are not toxic, the silica shell can prevent the dyes from photobleaching, they are highly hydrophilic, and they are photophysically inert since they are not involved in the abosorbance and emission processes.116 The preparation of the first dye-doped silica nanoparticles (DDSNs) was developed by Van Blaaderen and co-workers in the 1990s.117,118 This method, termed the Stöber-method, uses monomeric tetraethoxysilane (TEOS) as a precursor to the silica and organic dyes are covalently linked to the highly porous silica matrix. This method has high yield and is low cost and eco-friendly. Another recently developed method that is well-accepted by researchers involves synthesis in reverse microemulsions. Nanodroplets of water surrounded by a surfactant are dispersed into a continuous organic solvent. This method offers relatively easier control over the size of the silica NPs.120 Bonacchi et al. recently reviewed the synthesis and characterization of DDSNs.116

The optical properties of the DDSNs are controlled by the amount and properties of the molecular dyes inside. Shi and coworkers described pH nanosensing with DDSNs that were synthesized by a reverse microemulsion method and doped with a pH sensitive squaraine dye.120 The authors modified the surface of the NPs with carboxyl groups to avoid aggregation of the particles and increase water solubility. They optimized the amount of dye molecules doped inside the silica particles for optimal sensitivity while also decreasing self-quenching. They discovered that 224−272 nmol of squaraine dye molecules per mg of NPs provides the highest sensitivity. The intensity of the emission spectra increased linearly with increasing pH from 3.01 to 5.72. Many other ions, including alkali, alkaline earth, and transitional metal ions do not significantly interfere with the performance of this pH sensor. Since hydrophilic DDSNs exhibit excellent biocompatibility, they have accordingly found use in biological sensing and imaging. Wang et al.121 demonstrated the labeling of glycan by DDSNs. The authors also reported using DDSN labeled glycan to image and detect bacteria and to study glycan−lectin interactions on a lectin microarray. Fluorescein (FITC)-doped silica nanoparticles (FSNPs) ∼100 nm in diameter were synthesized by a modified Stöber-method and functionalized with perfluorophenyl azide (PFPA) silane because PFPA can be photochemically or thermally activated to singlet perfluorophenylnitrene, which forms covalent adducts with neighboring molecules by CH insertion and/or a CC addition reaction. PFPA-FSNP was labeled with different carbohydrates by incubating in microvials with UV irradiation for 10 min. The resulting solution became highly fluorescent. They tested the activity of the FSNP-carbohydrates (FSNP-mannose and FSNP-galactose) by treating them with Con A, which specifically binds with mannose. This results in the agglomeration of the NPs and a corresponding decrease in FL intensity of the NPs. Since Con A does not bind with galactose, the emission spectra did not change. The authors applied these FSNPs to the biological imaging and detection of E. coli. They treated the FSNP-mannose with two bacteria strain; one had a mannose specific binding domain while the other did not. The first sample showed a strong FL intensity while the second did not exhibit FL. They also studied the carbohydrate−lectin interaction on a lectin microarray, using this glycan label. The regions with Con A showed 10 times higher FL intensity than those treated with a nonmannose binding lectin. This glycan labeling method is simple and effective, and the labeled glycan maintains its specificity and reactivity. Carbon Nanoparticles. Carbon based nanomaterials are drawing more and more attention for sensing based applications because these nanomaterials are chemically inert and have low cytotoxicity, high biocompatibility, and unique electronic properties.122,123 Examples include carbon nanotubes, graphite, graphene oxide, and carbon NPs. In this section, we focus on the recent FL-based sensing applications of carbon nanoparticles (CNP). There are several approaches for synthesizing CNPs. CNPs can be prepared by thermal decomposition of organic molecules at high temperature.124 The as-prepared CNPs usually have fairly large sizes.125 There are several methods to prepare CNPs from graphite, including electro-oxidation of graphite126 and laser ablation of graphite.127 These methods are complicated, and it is difficult to synthesize CNPs in large quantity. Recently, several research groups reported the preparation of CNPs from cheap and easily accessible material, 556



Review

“Turn Off” Sensor. Quantum Dots. Mercaptoacetic acid (MAA)-modified ZnSe QDs detected Cu2+ and Ni2+ ions through the “turn-off” mode.136 MAA modification rendered the QDs hydrophilic, suitable for the detection of heavy metal ions in aqueous solution. The presence of Cu2+ or Ni2+ ions quenched the FL of the QDs. The FL intensity therefore decreased as a function of the metal ion concentration. The QDs selectively detected Cu 2+ and Ni 2+ ions in the concentration range of 140−2000 and 10−1000 μg/L, respectively. The FL of the QDs is not only quenched through energy transfer but also can be quenched via electron transfer (ET), when electron acceptors are in close proximity. Since there is no overlap of the emission spectrum of the donor and acceptor, the quenching of the FL of QDs is not due to the FRET process. There is an increasing number of sensing applications based on the QD-ET process. QD/dopamine bioconjugates were reported to sense in vitro and intracellular pH by an ET process.137 In this paper, a dopamine labeled peptide was attached to a QD through a (His)6 sequence. In acidic conditions, the reduced form of the dopamine (hydroquinone) is a poor electron acceptor and intense FL of the QD occurred. With increasing pH, the hydroquinone becomes oxidized by O2 to the strong electron acceptor (quinone), which can accept electrons from the QDs and quench the FL. When the ratio between the dopamine and QD is constant, the FL intensity (or the quenching efficiency) is directly proportional to the amount of quinone, which depends on the pH of solution. Most importantly, in the range of pH 7−11.5, the quenching response of QDs was linear. Further, they extended the application to intracellular pH sensing. They injected the pH 6.5 conjugate solution into cells and monitored the FL intensity changes at different times. At 60 min, the pH measured by the sensor was 12.8, which is only 10% higher than the expected value of 11.5. The authors also compared the pH of single cells with the average and found excellent agreement, which indicated that single cell resolution is possible. It is not possible that real biological samples have such a broad range of pH; however, this method enables sensing of intracellular pH by QD-based ET. Metal Nanoparticles. Ag nanoclusters (NCs) are of great interest as they are excellent fluorophores for optical sensing, biolabeling, and single molecule microscopy. Zhu and coworkers synthesized Ag NCs using a microwave assisted green synthesis with polymethacrylic acid as a template.138 With this approach, uniform and monodisperse Ag NCs were formed as microwave irradiation offered rapid and uniform heating of the solution for homogeneous nucleation and shorter crystallization times. These Ag NCs were stable for a month when stored in the dark at room temperature. The authors studied the effect of pH and Ag+ concentration on the synthesis of the Ag NCs. The fluorescence of the Ag NCs was quenched in the presence of Cr3+. The Ag NCs provided a 28 nM limit of detection that was highly selective to Cr3+ over other metal ions. Ying and co-workers synthesized FL Au NCs by a protein templated method and used them for Hg2+ detection.139 In the presence of Hg2+, the FL emitted by Au NCs was quenched due to the metallophilic interactions between Hg2+ and surface Au+. Hg0 has a weaker binding energy with Au+. Thus, by adding the strong reducing agent sodium borohydride, the authors recovered the FL of the Au NCs by reducing Hg2+ to Hg0. The size of Au NCs did not change by the addition of Hg2+, which ruled out Au NC aggregation as a FL quenching

such as candle soot. Mao and co-workers demonstrated the use of nitric acid to oxidize carbon soot and separate the CNPs using gel electrophoresis.128 Ray et al. later modified this and separated the CNPs by repeated centrifugation in a solvent mixture.129 CNPs prepared by this method were used as the FRET acceptor for sensing of Ag+,130 Hg2+,131 nuclei acid,132 and thrombin.132,133 Graphite-based materials are known as “super-quenchers”, acting as the acceptor in a FRET process, because of their much higher quenching efficiency compared to molecular dyes. The sp2 electronic hybrid structure and large conjugate plane gives graphene strong electron capturing ability.134 CNPs with the same sp2 electronic structure showed similar quenching properties.133 Li et al. reported the use of CNPs prepared from candle soot for FRET-based detection of Ag+ in aqueous solutions, which gives a lower detection limit compared to singlewalled carbon nanotubes and graphene oxide.130 In this method, CNPs were directly prepared by lighting the carbon soot and suspending in a water/ethanol (1:1) solution by sonication. The solution was centrifuged to separate out large soot particles and the supernatant collected and recentrifuged to get a black precipitate product. The precipitate then was dispersed in a (1:1) water−ethanol mixture. This cheap and simple method produces CNPs with diameters ranging from 25 to 40 nm, which is desirable for FL based applications. CNPs that are too large precipitate easily and those that are too small (below 10 nm) exhibit photoluminescence emission that can interfere with analyte detection.135 The CNPs served as the acceptor in the FRET based detection of Ag+. Fluorescent dye (ROX) labeled cysteine (C) rich ssDNA served as the FRET donor. Ag+ has a strong and specific interaction with cysteine, which reshapes the ssDNA to a hairpin structure. When there is no Ag+ in the sensing system, π−π stacking interactions lead to the adsorption of the DNA on the CNPs, which quenches the FL emission of the dye. The addition of Ag+ recovers the FL signal of the dye molecule as the hairpin structure forms and desorbs from the CNPs. The FL was excited at 580 nm and the intensity measured at 601 nm for different Ag+ concentrations. The detection limit of Ag+ was 500 pM, which is much lower than that on single walled carbon nanotubes (SWCNTs) and graphene oxide (GO). The authors speculated that the spherical morphology and unsmooth surface of the CNPs contributed to the enhanced sensing. This method showed high specificity to Ag+. The same group demonstrated the detection of Hg2+ in aqueous solution with a detection limit of 10 nM using a similar strategy.131 On the basis of the superquenching property of CNPs, Pang and co-workers133 reported the first UCP-CNP FRET based biosensor for the detection of thrombin. They prepared the CNPs with the same method as shown previously. Without thrombin, the thrombin aptamer modified UCP comes into close proximity to the CNP through π−π stacking interactions between aptamer and CNP, leading to FRET-based quenching. In the presence of thrombin, the aptamer forms a quadruplex structure which has much lower π−π stacking interactions with the CNP, causing the UCP to diffuse away, thus restoring its emission. The FL intensity varied linearly with thrombin concentration in the range of 0.5−20 nM with a detection limit of 0.18 nM. The sensor showed high specificity toward thrombin in the presence of metal ions, amino acids, and proteins. The authors extended the sensing application to thrombin sensing in human serum and plasma with similar behavior. This sensing strategy is simple, sensitive, and can be generally applied for other biological analytes. 557



Review

allowing easy discrimination superior to those using molecular beacons. The approach is flexible and does not require modification of NPs, complicated instrumentation, or high temperature for operation. Huang and co-workers detected prion protein based on FL quenching by Au NPs through long-range resonance energy transfer from quantum dots (QDs) to Au NPs.144 First, carboxylic acid terminated QDs were functionalized with nitrilotriacetic acid (NTA) and Ni2+ which then bind to the prion protein (PrPc) through the N-terminal histidine tag. The FL of the QD becomes quenched by strong interaction between the prion protein on the QD and an antiprion aptamer bound to Au NPs. The interaction leads to formation of a PrPc−aptamer duplex, which brings the Au NP and QD together for FL quenching. The authors reported a detection limit of 33 aM for PrPc. Ratiometric Sensing. Dye-Doped Silica Nanoparticles. We discussed earlier pH sensors based on monitoring the FL intensity of DDSNs. Zhang and co-workers described FRETbased ratiometric pH sensing using dye pair doped mesoporous silica NPs.145 Fluorescein isothiocyanate (FITC) and rhodamine B isothiocyanate (RBITC) were codoped into the NP, which served as the FRET donor and acceptor, respectively. RBITC quenched the emission of the FITC (at 521 nm) and emitted a FRET signal at 575 nm. The emission signal intensity of FITC increased with increasing pH value while the intensity of the FRET signal remained the same. By monitoring the intensity ratio of IFITC/IRBITC as a function of pH with different ratios of the two dyes doped in the NPs, the authors discovered that the pH sensing range varied with different doping ratios and that the largest range from pH 4−9 occurred with a FITC− RBITC of 1:2. The detection sensitivity also varied with different doping ratios, where a FITC−RBITC of 4:1 was most sensitive. The authors attribute this to the increased interaction of H+ with FITC at high local concentrations. In the previous application, two different kinds of dyes were codoped inside silica NPs during the synthesis. Lu and coworkers146 instead covalently linked RBITC on the surface of silica NPs with FITC-doped in the interior. The FITC served as a reference dye and RBITC as a response dye. These NPs were also functionalized with polyethyleneimine (PEI) as a stabilizer and chelating agent for the detection of Cu2+ in water samples and cells. The NPs emitted at 518 and 580 nm for FITC and RBITC, respectively. The RBITC emission band decreased through quenching in the presence of Cu2+ due to its binding to the surface while the reference FITC was unaffected. The FL intensity ratio of the bands showed good linearity in the range of 1 × 10−8 to 1 × 10−6 M (1 × 10−7 to 8 × 10−7 M for real water sample) with a 10 nM detection limit and color changes observable by the naked eye. The response time was 20 s, and the NPs can be regenerated with EDTA. Other metal ions were tested. Ag+ and Hg2+ affected the FL intensity of the NPs. The addition of NaCl recovered the FL signal because Cl− precipates with Ag+ and forms complexes with Hg2+. Co2+ and Ni2+ can form complexes with PEI at high concentrations, which can be overcome by the addition of HNO3. Acidic conditions do not affect the response to Cu2+ significantly due to the high affinity of PEI to Cu2+. The authors imaged intracellular Cu2+ with confocal laser scanning microscopy by incubating HeLa cells in the nanoprobe solution (4 h at 37 °C) and exposing to Cu2+ for 15 min. Polymer Nanoparticles. Polymer NPs embedded with analyte specific molecular dye molecules are a class of NPs

mechanism. The Au NCs were stable in solutions containing different anions and buffer and highly selective to Hg2+ over other environmentally relevant metal ions. This group detected Hg2+ at a concentration as low as 0.5 nM. The approach was extended to a paper test strip system for rapid detection of Hg2+ since the FL could be observed by the naked eye. Irudayaraj and co-workers used denatured bovine serum albumin (BSA) for the synthesis of fluorescent Ag NCs for Hg2+ detection.140 Denatured BSA contains 35 thiol groups available to act as chelating groups to sequester Ag+ and passivate/stabilize the metallic surface of the Ag NCs. BSA denatured by using both guanidine and tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) stabilized the Ag clusters more efficiently than BSA denatured by either guanidine or TCEP alone. In the presence of Hg2+, FL quenching of the Ag NCs occurred due to the metallophilic interaction between Hg2+ and Au+ as described earlier. FL quenching was also observed in the presence of Cu2+. To improve the selectivity toward Hg2+, the authors added a 2,6-pyridine dicarboxylic acid chelating agent to mask the interference from other divalent metal ions. The probes were stable in high ionic strength 1 M NaCl solution and detected 10 nM Hg2+. Tseng and co-workers were the first to report CH3Hg+ and Hg2+ detection in seawater using lysozyme type VI-stabilized fluorescent Au NCs (Lys VI Au NCs or Au-631).141 Here Lys VI functioned both as a reducing and stabilizing agent. An increase in Lys VI concentration during synthesis led to an increase in quantum yield and FL intensity and a decrease in Au-631 size. Au-631 exhibited high stability in high concentrations of glutathione and NaCl, suggesting that they could be used in physiological conditions. FL quenching occurred in the presence of CH3Hg+ and Hg2+, attributed to the high affinity metallophilic Hg2+−Au+ interaction. The selectivity of Au-631 for Hg2+ is 500-fold over other metal ions. Compared to bovine serum albumin (BSA)-stabilized Au NCs, Au-631 NCs are more sensitive since they contain more Au+ on the surface to interact with Hg2+ ions, since BSA contains more tyrosine residues that can reduce Au+ through their phenolic rings. The limit of detection of these probes is 3 pM and 4 nM for Hg2+ and CH3CH+, respectively. Fluorescent Au/Ag core/shell NPs were synthesized using a synthetic FL dipeptide β-Ala-Trp as a reducing and stabilizing agent.142 Dipeptide is fluorescent. The Ag+ concentration controlled the shell thickness. These probes exhibited quenched FL in the presence of Hg2+ due to the formation of a complex between the free carboxylate groups of β-Ala-Trp and Hg2+ ions, leading to aggregation of the NPs. They achieved a 9 nM limit of detection in the presence of other divalent metal ions. The FL properties of the Au/Ag NPs could be easily recovered by removing Hg2+ ions through complexation with EDTA. Tan and co-workers used a combination of a DNA ligation reaction and the ability of Au NPs to quench FL for the detection of a single nucleotide polymorphism (SNP).143 This was based on the difference in the affinity of single and double stranded DNA to Au NPs. The target DNA (T1) formed duplexes with the probes (P1 and P2; one of them fluorescently labeled) by a ligation reaction. Upon addition of the Au NPs, the duplexes do not adsorb on to the NPs and a strong FL signal appears. In the presence of mismatched DNA (T2), the probes P1 and P2 are not linked by DNA ligase. Upon addition of Au NPs, FL quenching by the Au NPs occurs because they adsorb strongly to the Au. The mismatched T2 causes a 22% greater reduction in FL intensity compared to the target T1, 558



Review

intracellular pH, the authors labeled the NP with F3 peptide, which has a high affinity toward tumor cells. A volume of 9 L of rat glioma cells were studied in this work. The authors measured an intracellular pH value of 7.1 ± 0.2 (which is similar to the value that is measured using NMR spectroscopy) as compared to 6.3 ± 0.2 for the unmodified NPs in a different environment. The authors attribute the high signal-to-noise ratio in this study to the minimal autofluorescence (

Nanoparticles in Measurement Science - Analytical Chemistry (ACS

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